CA1256023A

CA1256023A - Method of radioactively labeling diagnostic and therapeutic agents containing a chelating group

Info

Publication number: CA1256023A
Application number: CA000472895A
Authority: CA
Inventors: Jannis Stavrianopoulos
Original assignee: Enzo Biochem Inc
Current assignee: Enzo Biochem Inc
Priority date: 1984-01-30
Filing date: 1985-01-25
Publication date: 1989-06-20
Also published as: EP0150844A3; ATE92190T1; JPS60178827A; AU597208B2; DK39985A; NO850353L; DE3587477T2; EP0150844A2; IL74187A0; AU3821385A; US4707352A; DK39985D0; DE3587477D1; US4767609A; US4772548A; IL74187A; ES539927A0; ES8704001A1; JPH0822822B2; EP0150844B1

Abstract

ABSTRACT OF THE DISCLOSURE
A method of forming a therapeutic or diagnostic agent labeled with a radioactive metal ion, which comprises: contacting an unlabeled therapeutic or diagnostic agent, consisting of a substantially non-metal chelating portion and a chelating portion capable of chelating with the radioactive metal ion, with an ion transfer material having the radioactive metal ion bound thereto and having a binding affinity for the radioactive metal less than the binding affinity of the chelating portion for the radioactive metal ion, wherein prior to contacting the chelating portion is unchelated or is chelated with a second metal having a binding affinity with the chelating portion less than the binding affinity of the radioactive metal ion, whereby a radiolabeled therapeutic or diagnostic agent is formed by the contacting, and separating the radiolabeled therapeutic or diagnostic agent from the ion transfer material, is disclosed along with various components and kits useful in practicing this method and several variations thereof.

Description

~,L2~6023 TITLB OF TE~ IN7B~TIO~
MET~OD OB ~ IOACTIVELY LABE~ING
DIAGNOSTIC AND THERAPEUTIC AGENTS
C~NTP~INING A C~ELATING GROUP

BAC~GRO~D OF TEB INV~N~IO~
Field of the Invention:
The present invention relates to methods of radioactively labeling diagnostic and therapeutic agents and is particularly related to systems in which a metal ion is bound to the labeled molecule through a chelating group.

Description of the Rrior Art:
The use of radioactively labeled diagnostic and therapeutic agents has become routine practice in clinical and analytical laboratories throughout-the world. Such radioactively labeled compounds are used both in vitro (for example, in radioimmunoassay sys-tems) and in vivo (for example, both in diagnostic imaging techniques and in radiation therapy techni-ques).
Initially, the number of radioisotopes that could be firmly attached to the typical organic molecules used as diagnostic and therapeutic agents was limit-ed. The difficulty in forming stable carbon-metal bonds prevented the early utilization of many~radio-active metals and typically limited radioisotopes used to label organic molecule~ to isotopes of phos-phorus, carbon, hydrogen, and iodine.
Recently, a new approa~h has enabled the la~eling of such asents with metal ions. In this approach, a chelating moiety is covaiently attached to the mole-cule of interest, and a radioactive ion is then *;

,~ ' ~ ~256023 h l chelated by the sequestering groups of the chelator.
The chelating moieties which have generally been used for this purpose in the prior art have been analogues or derivatives of ethylenediaminetetraacetic acid (EDTA3, although many variations have also occurred.
For example, in 1968, W. F. Benisek and F. M.-Richards suggested the covalent bonding of chelating groups based on methylpicotinimidate to the amino function of a protein molecule in order to facilitate crystallographic investigation of protein structure by binding a metal to the chelating site on the protein [J. ~iol Chem., 2~3 4267-4271 (1968)]. Like-wise, in U.S. Patent No. 4,043,998, the compound 1-(p-benzenediazonium)ethylenediaminetetraacetic acid, said to be a powerful chelating agent which can be bonded strongly to proteins through its diazonium group, was disclosed. In Science, ~09, 295-297 (1980), B. A. Khaw et al. disclosed the use of a bifunctional chelating agent, diethylenetriaminepen-taacetic acid (DTPA) to label an antibody with a radioactive isotope and the subsequent use of that labeled antibody to image experimental myocardial infarctions in dogs. ~,The metal binding erficiencies of the resulting compounds were low, however, ~ince attachment occurred through one of the carboxylate groups which would normally have participated in binding to the metal ion. Similarly, D. A.
Scheinberg and O. A. Gansow taught in Science, 215 1511-1513 (1982), the use of DTPA and EDTA analogs covalently bonded to antibodies to image mouse erythroid tumors.
Unfortunately, the radioactively labeled materials previously available suffered from several ~.256023 _ disadvantages. Thi~ was particularly true~ for imaging agents and other molecules labeled with an isotope of high specific activity. The short half-lives of the radioactive isotopes used and the radiation-induced degradation of the labeled mole-cules greatly reduced the shelf-lives of these materials and, when imaging agents are involved, greatly increased the amount of background radiation present. Furthermore, health hazards to the techni-cians handling these materials and hazards associated with disposing of the associated waste generated at various steps of synthesizing labeled compounds made the handling of radioactively labeled compounds difficult.
Typically, as disclosed by Scheinberg and Strand in the article cited aoove, a bifunctional chelate was coupled to a target molecule, after which any metal ions present were removed by dialysis, typi-cally against a solution containing low molecular weight chelating molecules such as EDTA. The chelate-conjugated molecules were then labeled with a radioactive metal solution, after which free metal was removed, for example, by ion-exchange chroma-trography. The resulting labeled product was then stored and later used in the diagnostic or the~ peu-tic process. Using such procedures, considerable handling of the radioactive material and generation of radioactive waste occurred, a disadvantage not overcome by any teachings of the prior art.

SC~MaRY 0~ TEE INV~NTIO~
The present invention provides a universal method which can be used to radioactively label any diagnos-tic or therapeutic agent having a ligand portion '.

~ ~2560~3 thereof which is cap~le of binding with a radioac-¦ tive metal ion. The labeling occurs immediately ¦ prior to the utilization of the agent and produces ¦ little or no radioactive waste.
¦The invention provides a method of radioactively ¦ labeling a diagnostic or therapeutic molecule with a radioactive metal ion, which comprises:
(A) contacting ¦(1) an unlabeled therapeutic or diagnostic ¦ agent comprising ¦(a) a substantially non-metal chela-¦ t ng portion attached to ¦(b) a chelating portion capable of ¦ substantially chelating with said radioactive metal ¦ ion, with ¦(2) an ion transfer material having said radioactive metal ion bound thereto and having a binding affinity ~of said chelating portion for said radioactive metal ion, wherein prior to said contacting said chelating portion is unchelated or is chelated with a second metal ion having a binding affinity with said chela-ting portion less than the binding affinity of said radioactive metal io~, whereby a radiolabeled ~hera-peutic or diagnostic agent is produced b ~ said contacting; and (B) separating said radiolabeled therapeutic or diagnostic agent from said ion transfer material.
Additionally, the labeling method described above can be used as the first step of a diagnostic or therapeutic prccess, after which the normal steps of the process are carried out in their usual rashion.

~i . ' `' ' .

~ ~LZ56023 Typical of such proce$ses are radioimmunoassay and ln vivo diagnostic and tnerapeutic techniques.
The invention provides, in addition to the afore-mentioned process, various elements and components to be used therein in the form of kits comprising these componenls and other components used in the various processes.
In essence, the invention is based on the dis-covery that, if conditions are properly selected, hazards involving radioactive waste and radioactive products can be ameliorated by utilizing an ion transfer process as the last step prior to the ulti-mate use of a therapeutic or diagnostic molecule having a radiolabel. Thus, the necessity of handling radioactive material during the preparation of a diagnostic or therapeutic molecule is avoided and no waste radioactivity is generated in the clinical or analytical laboratory environment. Uses for the pro-cess, system, and components of the present invention are unlimited and include all of the uses to which prior a~t techniaues involving radiolabeled diagnos-tic and therapeutic molecules have been put as well as other uses disclosed herein.

D~SCRIPTIO~ OF~ PRgEBRRED EMBODI~E~TS ~
The terms "therapeutic or diagnostic agent" as used in the specification and claims of this appli-cation includes any substance or substances either alone or in mixtures which, when labeled with a radioactive metal ion, can be used in the treatment of a disorder of an animal or human body, in an in vivo diagnostic technique involving a human or animal body, or in an in vitro diagnostic technique for any ~.25~0~3 analyte whose detection is desired. Typical oftherapeutic agents are radioactive drugs containing beta-emitting radionuclides which are used for therapeutic purposes. These agents localize in path-ological tissue and destroy it by ionizing radiation.
In vivo diagnostic agents typically incorporate a gamma-emitting nuclide which, because of the physical or metabolic properties of the molecularly recogniz-able portion of ~he agent, localizes in a specific organ after administration. Diagnostic images re-flecting organ structure and/or function can then be obtained by means of detection devices that detect the distribution of ionizing radiation emitted by the nuclide. In vitro diagnostic agents are exempli,ied by radioimmunoassay agents which are in wide-spread clinical use. These agents are employed in the measurement or minute quantities of various biologi-cal substances, such as hormones.
Diagnostic and-therapeutic agents of the inven-tion have two functionally different portions of the molecule or molecular conjugate ~although these may be at least in part the same structural portions in some molecules). These portions are ta) a substan-tially non-metal chelating portion attached to (b) a chelating portion c~apable of chelating with the radioactive metal ion being used. By ~substan~ially non-metal chelating portion~ is meant to include not only molecular portions which carry no metal-chelat-ing groups, but also molecular portions which may carry certain groups capable of metal chelation but which do so with substantially less affinity than portion (b) he ~helating portion.-. `

~256023 Particularly preffrred among the "substantiallynon metal chelating portions" (a) are those which are molecularly recognizable portions. The phrase "mole-cularly recognizable portion" denotes any molecular portion of the total molecule which is capabie of being recognized by a complementary system or mole-cule in the system in which the agent is being used.
Molecular recognition, as will be understood by those skilled in the art, includes the non-covalent bind ng in three dimensions between complementary portions of two molecules. A molecularly recognizable portion on an agent may be of low molecular weight (about less than MW 2,000) or of high molecular weight. For example, it can be a polynucleotide sequence, such as RNA or DNA, to be recognized by its complementary sequence; an antigen portion (e.g., a drug, a pesti-cide, a metabolite, a physiologically occurring compound), to be recognized by its corresponding monoclonal or polyclonal antibody; an antibody por-tion, to be recognized by its corresponding antigen;
a lectin portion, to be recognized by its sugar; a sugar portion; to be recognized by its lectin; a hor-mone portion, to be recognized by its receptor; a receptor portion, to be recognized by its hormone; an inhibitor portion, to be recognized by its enzy~e; an enzyme portion, to be recognized by its inhibitor; a cofactor portion, to be recognized by a cofactor enzyme binding site; a cofactor enzyme binding site portion-, to be recognized by its cofactor; a binding ligand, to be recognized by its substrate and vice versa (e.g., biotin-avidin); or any permutation or combination thereof. Among the most common molecu-larly recognizable portion~ are the three~dimensional .1 ' - ' ` " ~

3~256023 protein arrangements in aatigens and antibodies of f various sort~, the cell wall structures present in various cells, and the nucleic acid sequences present in the DNA and RNA of organisms. It is preferred in many circumstances that the molecularly recognizable portion be either a natural constituent of a biologi-cal system or recognizable by a natural constituent of a biological system. Thus, in a competi~ive radioimmunoassay using a solid phase antibody which binds to a natural constituent present in the serum of a human or animai, the molecularly recoqnizable portion preferably would have the same structural fsature~ present in the natural component with which it was in competition for binding with the antibody.
In a therapeutic agent designed to concentrate radio-activity in a specific tissue, the molecularly recognizable portion would be recognizable -by a natural component of that tissue. ~owever, it is the function of being molecularly recognizable that is important rather than the actual structure. For example, the molecularly recognizable portion could be an analog or an artifical component which binds more tightly than any natural component of a biolo-gical system and therefore is more selective for a particular tissue or other component of a biol~gical system. Additionally, both the molecularly recogniz-able portion and the component which recognizes this portion may be entirely artificial, particularly in an in vitro diagnostic assay. As used in this appli-cation, the phrase ~complementary substance" refers to the component which recognizes the molecularly recognizable portion of the agent, whether the ~256023 '_ g .~
~ ,, complementary substance is of artificial or biological origin.
Furthermore, the molecuiarly recognizable portion need be only a small part of the therapeutic or diag-nostic agent and further need not correspond to an entire molecule present in any system. For example, when the ~olecularly reco~nizable portion is pro-teinaceous, it may be a relatively short sequence of amino acids ~ound within a much larger sequence of amino acids as would be typical for a hapten or bind-ing site which formed part of a large protein.
The second essential portion of the agent is the "chelating portion. n Chelates are coordination complexes that are formed between a metal ion and a ; ligand that contains at least two electron-donating groups arranged so that a ring structure is formed upon coordination. Especially stable are chelates containing 5- or 6-membered rings. Typical func-tional groups involved in chelation include acidic or anionic groups derived from carboxylic acids, oximes, hydroxyl compounds, phenols, sulfonic acids, and mer-captans. Uncharged functional groups capable of i being involved in chelation include amines (primary, secondary and tertiary)~ carbonyl groups, thiocar-bonyl groups, nitroso groups, and cyclic amines, such as those typically present in heterocyclic compounds.
A ligand involved in complexation can be either charged or uncharged.
The chelating portion of the agent typically will be formed by reacting a derivative of a known chela-ting agent with a molecule having a portion that forms the substantially non-metal chelating portion~
of the final therapeutic or diagnostic agent.

.

~A` ¦ Preferred are chelating portions which comprise a I diamine wherein the two amine grsups are substit~ted ¦ with two acetic acid moieties, with the two amino groups and/or the four acetic acid groups being ¦ capable of donating an electron pair to the same l metal ion. Typicalls~, the amino groups will be ¦ covalently attached to adjacent carbon atoms.
¦ Preferred are derivativec of ethylenediaminetetra-, I acetic a~id and other chelating groups having a binding constant for any radioactive metal ion at least as great as that of EDTA for the same metal ion. The ethylenediaminetetraacetic acid derivative 1,2-diaminocyclohexaneacetic acid and its derivativec ¦ and analogs are especially preferred By derivatives and analogs is meant compounds having the basic ¦ skeletal structure and functional groups of these ¦ compoundq but having additional functional groups ¦ which do not prevent the resulting compounds from ¦ functioning as chelatlng groups. Typical chelating ¦ molecules which can be modified to form the chelating ¦ portion of the agent are DCTA, EDTA, tartaric acid, alpha-benzoin oxime, 1,10-phenanthroline, and similar ¦ well known compounds.
¦ The substantially non-metal chelating portion of I the molecule may be derived from any molecule of ¦ small or high molecular weight, any molecular com-¦ plex, or any biological system te.g., a virus, a cell ¦ or a group of cells). Among the common molecules ¦ which may be used as sources are amino acids, sac-¦ charides, nucleotides, proteins, polysaccharides, ¦ lipopolysaccharides, protein complexes, single- or ¦ double-stranded nucleic acids or segments thereof, whole viruses or viral compounds such as cores or Il . `
.

~ 1256023 _apsids, bacteria, tissue cells, and the like. A~ong the most common proteins are the structural proteins, enzymes, immunoglobulin~, and fragmentQ thereo~.
Among the most common nucleic acids are DNA and RNA
of various type~, such as t~NA, mRNA, rRNA, and the like. Bacteria, either whole or fragments thereof, such as cell wall~ or other recognizable por~ions, include both gram positive and gram negative bac-teria. Fungi, algae, viruses and other microorganisms (and fragment~ thereof) are also included as well as animal ~e.g., mammalian) cells including red blood cells.
Because the principal aspect of the present invention contemplate~ labeling a preformed thera-peutic or diagnostic molecule con~isting of a non-chela'ing portion and a chelating por~ion, the generai technique~ of producing such moleculeq are not considered part of the presen~ invention, a}though certain types of chelating groups and methods of attaching th~m to the non-chelating pcr-tion of agents are discussed in later sections for purposes of illustration.
As discussed in the section of this application entitled prior art, many therapeutic and diagnostic agents having chelat~ng portionq and non-che~ating portion~ are already ~nown. For example, ~natowich et al., Science, 220, 613-61~ (1983), disclose a method of covalently coupling the chelator diethylene-triaminepentacetic acid (DPTA) to proteins, such as immu-noglobulins. Generally, a dianhydride of DTPA is reacted with a molecularly recognizable protein under straightforward conditions. This method may be used ~,~",, to attach a ligand to any molecule having an amino or hydro~yl group or a s~milar nucleophilic group. Since many molecules already contain one of these groups (and the remainder can generally be easily modified so that they do), this provides a general method of attaching a chelating group to any molecule of inter-est. Many similar methods, such as those disclosed in the references cited in the section of this applica-tion entitled "Description of the Prior Art,~ all of which are herein incorporated by reference, disclose further ligands and methods of modifying other mole-cules with them.
In addition to those agents previously known to the prior art, many other diagnostic and therapeutic agents having a molecularly recognizabie portion and a chelating portion can be synthesized by standard techr.iques of organic chemistry. For example, al-though the prior art has dealt with the attachmer.t of chelating groups to proteins, it is also possible to attach chelating groups to non-proteinaceous mole-cules of interest, such as lipids, hormones, and sugars Althou~n chelating groups have not previous-ly been attached to such molecules, many derivatives ~ of these various classes of biological compounds are i~ known which have cova~ent bonds formed through ~ car-bon, oxygen, nitrogen or sulfur atom to an organic radical not normally part of the compound. Minor variations of the techniques used to snythesize these known compounds can be used to attach chelating groups tG the recognizable molecules.
Likewise, chelating molecules can be modified by standard chemical techniques to provide a functional group through which attachment to the recognizable 1: i560Zl ~ -13-., molecule can take place. Several procedures~are disclosed for the chelating groups that have been previously modified for attaching to proteins, as has been previously discussed. Furthermore, since many chelating molecules contain at least one radical derived from acetic acid, these molecules can easily be modified using standard techniques to create a functional group on the alpha carbon through which attachment can take place to recognizable molecule.
The properly functionalized recognizable molecules and chelating groups can easily be attached one to the other by standard reactions of organic chemistry although, naturally, all the resulting compounds will not fall into the class of agents which exhibit the most preferred binding affinity.
Chelating groups that are analogs of 1,2-diamino-cyclohexaneacetic acid are particularly preferred for use in the practice of this invention. The chelating group is covalently bonded, generally though not necessarily through an appropriate bridging entity, to a diagnostic or therapeutic molecule of interest to crea'e agents useful in the practice of the in-vention. The chelating portion provides a strong bonding site for metal ions and, by selecting the proper linking structure, can be coupled-to a v~iety of sites on a wide range of molecules.
One advantage of 1,2-diaminocyclohexaneacetic acid analogs is that they can be successfully used with polynucleotides and nucleic acids, unlike cer-tain prior art aromatic chelating groups which cannot usually be used with polynucleotides because of intercalation. The cyclohexane-based dicyclohexane-tetraacetic acid (DCTA) analogs senerally do not ' I
, , . I

~256023 I interfere with any nor~al rea^tions of labeled joly nucleotides or nucleic acids and can additionally be used with any of the other molecularly recognizable portions discloqed herein. The DCTA analogs also have binding affinities for metal ions several orders of magnitude higher than those of EDTA.
Examples of therapeutic and diagnostic agents useful in this invention are also disclosed and dis-cussed in co-pending Canadian application Serial Number 472,862 by Y. Stavrianopolous, filed on January 25, 1985 and entitled "Detectable Molecules, Method of Prepara-tion and Use," which is assigned to Enzo Biochem, Inc.

The chemical structure of the preferred chelating gro~ps in a diagnostic or therapeutic agent a~
described herein is exemplified by the following structur~l formula:

Rl . R-A~N--C~2-COOM
--CH2COO~I

wherein R iS the substantially non-metal chelating portion of the therapeutic or diagnostic agent, Rl is Cl-C4 alkyl or is -C~2COOM, M is ~ or a cationic metal or a negative charge, and A is either a direct covalent bond or a b~idging entity such as, e.g., of the type shown in the aforementioned co-pending ap-plic o=. Since splcing is the main consideration ~ ~25~0~3 i I -15-,' I ~
rather than the structure Or the bridging entity, the chemical structure of the bridging entity is unim-portant and is not limited as long as -- among other things -- molecular recognition is not unduly hindered.
It is preferred to use a bridging entity to join the non-chelating molecule to the chelating molecule from which the chelating portion is derived. The selection of the bridging entity is, of course, varied depending on the type of moleculeQ involved, the number and nature of the available bonding site , the types of reactions which the labeled agent i5 to undergo, and other factors known to those skilled in the art. The linking group can be tailored to specific types of agents, for example, nucleotideQ, proteins, amino acids, enzymes, etc., to suit the needs of particular detection, imaging, or thera-peutic techniqueQ.
Examples of generally useful linking groups include beta-thiopropionic acid hydrazide, beta-thioethylamine, and isothiocyanate. In particular, beta-thiopropionic acid hydrazide has been found to . be highly suitable for the attachment of chelating ;~ groups to amine-containing molecules under ~;mild conditions. The preferred bridging entity for a particular non-chelating molecule depends on the reactive functional groups present in that molecule.
For example, molecules having a free amino group (such as proteins and peptides having one or more lysine residues) can be reacted with a carbonyl azide to form a peptide bond. Molecules having a free hydroxyl group (such as proteins haYing a tyrosine residue) can be reacted with an isothiocyanate or can I ~2S6023 be heated in the presence of the azid~ (which rear-ranges to form an isocyanate) to form a thiourethane or uzethane. Molecules having a carbonyl group can form a Schiff base with an amino group of a modified chelating molecule which can then be reduced if de-sired to a secondary anine. Many variation3 of these bonding technigues exist and may be used as deemed appropriate.
Examples of agent~ which can be used in the practice of the invention include tho~e in which a molecularly recognizable portion i~ derived from a nucleotide or related compound. Methods of forming such compounds are described in detail and claimed in copending Canadian application Serial Number 430,882, filed June ~, 1983, which 1s assigned to ~nzo Biochem, Inc. Accordingly, agenta whos~ molecularly recognizable portion i5 derived from DNA, RNA, a nucleotide, a deoxynucleotide, nucieoside, or a deoxynucleoside can easily be prepared using the methods described th~rein for modifying the nucleo-tide or related molecule, together with the methods described herein for coupling to chelating groups.
The ratio of tne non-chelating portion of the agent to the chelating portion need not necessarily be 1:1. There may be many more chelating por,tions than non-chelating portions, or vice versa. In the c-se when the ratio of chelating portions to non-chelating portiona is greater than 1, for example, 5-10 to 1 or even greater, the system amplifies the radiation provided by the primary recognition event by a factor equal to the ratio.

,~

.. ~256023 It should again be noted that the aspect of the present invention relating to labeling an agent already containing z chelating portion in no way depends upon the structure of the molecules being manipul ted b,ut rather depends on their chelating ability and their ability to be recognized on a mole-cular scale in a biological or biochemical syste~.. So long as chelation with radioactive metal ion is possible, molecular recognition can take place, and an ion transfer material is available which ha~ a lower binding affinity for the radioactive metal than does the chelating portion of the agent, the inven-tion can be practiced regardless of the structure of the molecule.
Likewise, the structure of the ion-transfer material is unimportant so long as the binding af-finity (i.e., che binding function) is within the limitations disclosed. Although, generally speaXing, it is sufficient for the practice of this invention to use an ion transfer material whose binding affin-ity for the radioactive metal ion is merely less than the binding affinity of the therapeutic or diagnostic agent for the same ion, it is preferred that the ratio of binding affinities be less than 0.1, more preferably less than 0.01, and most preferably less than 0.001, in order to ensure effective transfer of the radioac'ive metal ion from the ion transfer material to the agent.
Suitable ion transfer materials include both inorganic and synthetic organic products. Inorganic ion transfer mzterials include both the naturally occurring materials te.g., mineral zeolites such as ,. ~' , ` ' ~256023 sodalite and clinoptilolite, the green sands, and clays such as the montmorillonite group), and synthe-tic products such as the gel zeolites, dehydroxide~
of polyvalent metals such as hydrated zirconium oxide, and the insoluble salts of polybasic acids with polyvalent metals such a zirconium phosphate.
Preferre~ ion transfer materials are the synthetic organic cation exchange resins. These include weak-acid, cation-exchange resins and strong-acid resins. The weak-acid resins are generally based on acrylic or methacrylic acid that has been crosslinked with a difunctional vinyl monomer, such as divinyl benzene. Other weak-acid groups, such as phenolic or phosphonic functional groups, may also be used. The weak-acid resins are generally used at a p~ above 4.
The strong-acid resins are generally based on sulfo-nated copolymers of styrene and divinyl benzene.
These materials are particularly preferred because of their ability to exchange cations across the entire p~ range. The most preferred ion exchange materials are sufficiently porous to provide a large surface area on which exchange can take place. Pore sizes are preferably sufficient to allow easy passage of the agent through the pores and most preferably are several times the largest diameter of the molecule in question. ~owever, if the diagnostic or therapeutic agent i~ particularly large, transfer may occur on exterior surfaces only.
Many commercially available ion transfer materials are known and may be used in the practice of this invention if the guidelines set forth h~rein ~256023 -lg-.
are followed. For ~xample, Dowex 50* and materials having similar properties are partieularly suitable.
In general, the labeling process of the present invention is accomplished by contacting the thera-peutic or diagnostic agent as defined herein with an ion transfer material having the radloactive element bound thereto. The contacting may consist either of passing a solution containing the agent over a column of the ion transfer material or by ~uspending the ion transfer material in a solution of the agent.
Although these methods of contacting are preferred, any other method of intimately contacting a solution containing the agent with the ion transfer material is suitable. The amount of radioactivity bound to 1 the ion transfer material, the duration of the contact time, and the ratio of the amount of the diagnostic agent to the amount of the ion transfer material, as well as other conditions, vary depending on the amount of radioactivity needed for the parti-cular situation in which the agent i- to be used, as is well understood to those skilled in the art. If the conditions and contacting times are not known, they can easily be determined by simple experimen-tation. After a sufficient contacting time, the radioactivity labeled agent is separated from the ion transfer material by any suitable technique. Typi-cally, the ion transfer material will be present in the form of a column and the agent can be seoarated by elution. -Elution can occur using the solvent in which contacting took place, or a second eluent may be used if such treatment more easily dislodges the agent from the ion transfer material. If not already * Dowex 50 is a trademark.

, ~ Z56023 -20- ~
:' "~.-" knowr" suitable eluents may be determined by simple experimentation since elution of radioactivity i~
easily followed. It is particularly preferred that an eluent not permanently change a molecularly recog-nizable por'ion of the agent so that the~ recognition event can no longer t~ke place. However, a temporary change, for example in conformation, causes no harm if the recognizable structure can later be regained.
Thus elutions with solvents or solutions, or under conditions which result in a reversible conforma-tional change in the stxucture of a peptide, for example, are acceptable. Nevertheless, elutions of agents of biological origin at or near physiological conditions te.g., p~, ionic strength, temperature, etc.) is preferred, particularly if the eluent is to be directly in one of the diagnostic or therapeutic procedures which are latar discussed~
Any radioactive metal ion capable of producing a therapeutic or diagnostic result in a human or animal body or in an ln vitro diagnostic assay may be used in the practice of the present invention. Suitable ions including the following:

lZ56023 ~:' Antimony-124 Iodine-125 Scandium-44 Antim3ny~125 Iodine-131 Scandium-46 Arsenic-74 Iridium-192 Selenium-75 Iron-55 Silver-llOm Barium-103 Iron-59 Silver-lll Barium-140 Sodium-22 Beryilium-7 ~rypton-85 Strontium-85 Bismuth-206 Strontium-89 Bismuth-207 Lead-210 Strontium-90 Lutecium-177 Sulphur-35 Cadmium-lG9 Cadmium-115m Manganese-54 Tantalum-182 Calcium-45 Mercury-197 Technetium-99 Mercury-203 Tellurium-125m Cerium-139 Molybdenum-99 Tellurium-132 Cerium-14i Terbium-160 Cerium-144 Neodynium-147 Thallium-204 Cesium-137 - NeDtunium-237 Thorium-228 Chlorine-36 Ni~kel-63 Thorium-232 Chromium-51 Niobium-95 Thulium-170 Cobalt-56 Tin-113 Cobalt-57 Osmium-185+191 Titanium-44 Cobalt-58 Cobalt-60 Palladium-103 Tungsten-185 Erbium Platinum-19Sm Vanadlum-48 Europium-152 Praseodymium-143 Vanadium-49 Promethium-147 Gadolinium-153 Protactinium-233 Ytterbium-169 Gold-195 Yttrium-88 Gold-l99 Radium-226 Yttrium-90 Rhenium-186 Yttrium-91 ~afnium-175 Rubidium-86 ~afnium-175+181 Ruthenium-103 Zinc-65 Hafni~m-181 Ruthenium-106 Zirconium-95 - ;~256023 r ¦ The following non-limiting example illustrate-~
¦ the preparation of a diagnostic or therapeutic agent ¦ using a Dowex 50 column. The column i8 first equi-librated with a dilute solution of a buffer, for example, 0.05 M ammonium acetate and then loaded with a radioactive ion, for example, nickel-63, by pas~ing a solution of the ion through the column. After the column i~ prepared (when presented in kit form, as later described, the column would be prepared by one other than the ultimate user during preparation of the ~it), the agent having a chelating portion $s pAssed through the column and eluted a~ the radiola-beled metal chelato.
The labelinq procedure described above i3 particularly useful in combination with established therapeutic and d~agnostic techniques which use an agent ha~ing the properties described in this appli-cation. For example, a diagnostic agent u~eful in radioimmunoassay (R'A) can be labeled immediately prior to its use, thus greatly reducing non-3pecific binding caused by radiation damage which would occur with an agent which h~d been labeled and stored for a long period of time. RIA is a well-known technique and will not be descr~bed in detail here. For parti-culars, reference i~ made to Chard, "An Introduction to Radioimmunoa~say and Related Techniques,~ North-~olland Publishing Company, 1978.
Any of the many varia-tions of RIA ca~ be used, such a~ homogeneous phase RIA, heterogeneous or solid phase RIA, ~ingle anti-body or double antibody methods, and direct (forward) or reverse sandwich a~says. Particularly preferred ~ . `

~.25~023 -23- ' are solid phase systems wherein the antibody (IgG or IgM) is covalently coupled to an insoluble support so that both the antibody and the bound complex after incubation can be readily separated from the soluble free fraction. A wide variety of solid phase sup-ports have been described, which include particles of dextran or cellulose, continuous surfaces such as polystyrene or polypropylene discs, walls of plastic tubes, glass discs, glass particles, and the like.
Particulate solid phases are widely used for a variety of different assays and can be used in the practice of the present invention~ Antibodies are attached to the particles by any of a number of tech-niques designed to yield a non-reversibls covalent or non-covalent link between protein and particle, for example, directly or by cyanogen bromide activa.ion.
Other alternatives are the use of antibodies entrap-ped in the interstices of a polyacrylamide gel or bound to magnetic particles. An assay tube is set up containing either sample or standard, along with the tracer and an appropriate amount of solid phase bound antibody, plus a detergent to prevent aggregation of the particles and non-specific absorption of the tracer. After an incubation period during which the tubes are continuously mixed, the solid phase is sedimented by centrifugation; the supernatant is removed and the solid phase subject to two or more washes with buffer in order to remove free tracer trapped within and between the particles. The counts on the solid phase (bound fraction) are then mea-sured. Immunoradiometric assays, as described in Cherd at ps 423, can also be used. When a second ~ ' ~256023 -24- ~

antibody is used, the second antibody can be either IgM or IgG. The present invention is not limited to any of these techniques in particular.
Similarly, the method can be applied in vivo diagnostic and therapeutic techniques by labeling the agent immediately prior to its use. This aspect of the invention is especially important because of the high levels of radioactivity associated with such agents, especially therapeutic agents, which result in rapid degradation of any molecularly recognizable portion of the molecules and loss of specificity. By using the technique of this invention in combination with established in vivo techniques for using radio-active agents, destruction of any molecularly recog-nizable portion of the agent, which reacts with a complementary substance in a human or animal body to cause selective localization in a target region, is greatly reduced. Accordingly, it is possible in many cases to use a lower total mount of the radioactive isotope in a diagnostic technique because of increas-ed specificity. This technique is particularly suited to use with monoclonal antibodies to which a chelating group is attached.
The present invention lends itself readily to the preparation of kits comprising one or more of the elements necessary to perform the labeling process.
Thus, a kit may comprise a carrier being compartmen-talized to receive in close confinement therein one or more container means or series of container means such as test tubes, vials, flasks, bottles, syringes, or the like. A first of said container means or series of ntainer mean~ may contain the therapeutic .`

! ' 12560Z3 or diagnostic agent as described herein. A second container means or series of container means may con-tain aD ion transfer material capable of binding the radioactive metal ion of interest for the particular application of interest. Two embodiments for the second container means are possible with regard to the radioactive metal itself. In one embodiment, the ion is Dound to the ion transfer material during the process of manufacturing the kit. The user of such a kit is therefore not required to handle radioactive material in fluid form at any point prior to obtain-ing the diagnostic or therapeutic a~ent in the eluting fluid, which can be chosen so that it i8 immediately useable. Alternatively, the kit may provide an ion transfer material not having any radioactive metal ion bound thereto. This greatly simplifies preparation, storage, and handling of the kit itself. The radioactive metal ion is then bound to the ion transfer material by the user of the kit.
The ion transfer material may then be utilized to label several doses or aliquots of the therapeutic or diagnostic agent. Such a kit and procedure is parti-cularly suited for isotopes of very short life.imes, such as are often used in in vlvo procedures. Medi-cal technicians who would normally use solution chemistry to label a therapeutic agent comprising a chelating portion and an antibody, for example, can accomplish the same result using the techniques of this invention and a kit adapted to that use with less waste radioactivity and contaminated glassware.
It is preferred that the second container means be fitted with fluid inlet and outlet means whereby ,.`

~Z56023 the agent (unlabeled with radioactivity), when in-serted into the inlet means, intimately contacts the ion transfer material while passing through or being contained within said container means prior to exit-ing through the outlet means. It is particularly p-eferred that the inlet and outlet means be fitted with confining means, such as a screen, which prevent the exit of the ion transfer material from the con-tainer. In a particularly preferred embodiment o.
the present invention, the second container means containing the ion transfer material having the radioactive ion bound thereto is columnar or tubular in form, with the inlet and outlet means being at opposite ends of the tube. Thus, a user can ea~ily label any dianostic or therpeutic agent having a che-lating portion thereon by adding the agent through the inlet means and removing the agent as it exits the outlet means. Typically, passage of the agent through the container means would occur in solution, whereby the agent would intimately contact ion trans-fer material therein. The radiolabeled agent can be recovered either by force of pressure or suction or by allowing it to drain from the lower exit means or by passing an eluting fluid through the column, as is well understood by tho~e s~illed in the art. One suitable technique would be to use a disposable sy-ringe or other administering means suitable for use in the diagnostic or therapeutic procedure for which a radioactive agent is desired which is fiited with connecting means by which it can be attached to the exit means of the ion transfer material container.
The agent can then be withdrawn into the syringe with_, minimum danger of loss or contamination. Typically, '~

~256023 the kit would also contain a third container means having therein an eluant suitable for eluting the agent from the column. If the kit is intended for a particular in vitro diagnostic technique, for exam-ple, a competitive radioimmunoassay procedure, a fourth container means can contain a complementary substance capable of binding with any molecularly recognizable portion or the agent, for example, a solid phase antibody capable of binding both with the analyte and the diagnostic agent. If the unlabeled agent is present in a dry form (e.g., lyophilized), a fifth containing means containing a solvent may be supplied. A typical complete kit of the invention will contain at least the first two container means and associated substances and may optionally contain any other related materials useful for the procedure under consideration.

!~

Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A method of labeling a therapeutic or diagnostic agent with a radioactive metal ion, comprising:
contacting (1) an unlabeled agent, comprising (a) a molecularly recognizable portion attached to (b) a chelating portion capable of substantially chelating with, and having a binding affinity for, said radioactive metal ion, wherein said chelating portion is not a part of said molecularly recog-nizable portion, with (2) an ion transfer material having said radioactive metal ion bound thereto and having a binding affinity for said metal ion less than the binding affinity of said chelating portion for said radioactive metal ion;
forming a radiolabeled therapeutic or diagnostic agent by transfer of said metal ion from association with said ion transfer material to asso-ciation with said agent;
separating said radiolabeled thera-peutic or diagnostic agent from said ion transfer material.

2. The method according to Claim 1, characterized in that prior to said contacting step, said chelating portion is unchelated.

3. The method according to Claim 1, characterized in that prior to said contacting step, said chelating portion is associated with a second metal ion, said portion having a binding affinity for said second ion less than the binding affinity of said portion for said radioactive metal ion.

4. The method according to Claim 1, characterized in that the molecularly recognizable portion of said agent is selected from the group consisting of a natural constituent of a biological system, a protein-containing constituent, a nucleic-acid containing constituent, a saccharide-containing constituent, a hormone-containing constituent, an antigen, an antibody, a hormone receptor, a virus, a viral component, a bacteria, a bacterial component, a cell and a cellular component.

5. The method according to Claim 1, characterized in that said chelating portion com-prises a diamine having four acetic acid moieties attached to the two amino groups of said diamine.

6. The method according to Claim 5, characterized in that said chelating portion is a derivative or analog of ethylenediaminetetraacetic acid or trans-1,2-diaminocyclohexaneacetic acid.

7. The method according to Claim 6, characterized in that said chelating portion com-prises a radical having the formula wherein R1 is CH2-COOM or a C1-C4 alkyl and M is hydrogen, a cationic metal or a negative charge.

8. The method according to Claim 1, characterized in that the fraction of the binding affinity of said ion transfer material for said radioactive metal ion compared to the binding affinity of said chelating portion for said radioactive ion is less than 0.1.

9. An improved radioimmunoassay method comprising the steps of:
forming a diagnostic agent labeled with a radioactive metal ion according to the method of Claim 1, said molecularly recognizable portion being capable of participating in an antigen-antibody binding reaction; and employing said agent in a radioimmuno-assay procedure.

10. A kit suitable for forming a thera-peutic or diagnostic agent labeled with a radioactive metal ion, which comprises:
carrier means compartmentalized to receive in close confinement therein one or more containers;
a first container confined within said carrier and containing a therapeutic or diag-nostic agent comprising a molecularly recognizable portion attached to a chelating portion capable of chelating with and having a binding affinity for, said radioactive metal ion; and a second container confined within said carrier and containing an ion transfer material having said radioactive metal ion bound thereto, and having a binding affinity for said radioactive.metal ion less than the binding affinity of said chelating portion therefor.

11. The kit according to Claim 10 wherein the molecularly recognizable portion is chosen or prepared so as to be complementary to a substance in a particular tissue or region of a human or animal body.